Unique among the generalized class of lysosomal disorders are those that are inherited as a single gene defect but which are phenotypically characterized by deficiencies in the activities of multiple lysosomal hydrolases. Within this category are two related diseases, I-cell disease and pseudo-Hurler polydystrophy which are now called I-cell Type 1 and I-Cell Type 2, respectively. Cultured fibroblasts from these patients demonstrate multiple lysosomal hydrolase activity deficiencies while concomitant increases of these enzyme activities are found in the fibroblast media. The genetic lesion responsible for the mislocalization of the apparently unrelated lysosomal hydrolases is currently unknown, but evidence from our laboratory and others suggests that the genetic defect results in an altered oligosaccharide side chain on the I-cell acid hydrolases. The overall objective of the proposed research is to delineate the nature of the molecular defect at the protein level that is responsible for I-cell Type 1 and I-cell Type 2, while contributing valuable information concerning the role of protein-bound carbohydrates in the cellular localization and transport of lysosomal hydrolases in normal and disease states. The relationship of the altered carbohydrate structures on the I-cell acid hydrolases to their mislocalization will be examined by the use of subcellular fractionation. Normal, I-cell Type 1 and Type 2 non-labeled and pulse-labeled cultured fibroblasts will be subjected to carrier free continuous deflection electrophoresis, a technique which allows reproducible separation of different cell organelles. The results of these studies will yield information concerning the structural requirements for lysosomal localization and/or extracellular secretion of acid hydrolases. A portion of these studies will involve additional experimentation using the antibiotic Tunicamycin. These results should expand our preliminary observations on the role of the N-glycosidically linked oligosaccharides in the normal lysosomal localization of acid hydrolases and the mislocalization of the same enzymes to the extracellular milieu in I-cell Type 1 and Type 2.